PROJECT SUMMARY/ABSTRACT This project will expand the acquisition, reconstruction, analysis, and dissemination of 3D electron microscopic (3D EM) reference data, disclosing key ultrastructural details preserved within a remarkable collection of legacy biopsy brain samples from patients suffering from Alzheimer?s Disease (AD). These samples were originally collected, characterized and archived by neuropathologists R.D. Terry and N. Gonatas (at A. Einstein in the 1960?s), with later samples taken as part of a cerebrospinal fluid (CSF) drug infusion study involving S. Mirra (at Emory in the 1980?s). They were re-examined by Ellisman, Masliah, Terry, and Mirra in the 1980s, using early 3D EM methods, and were found to manifest excellent preservation of ultrastructure, showing paired helical filaments (PHF) and amyloid accumulations as well as modifications to subcellular organelles and cytoskeletons of the cell bodies, axonal and dendritic processes. Here, we will exploit recent advances in high throughput, automated 3D EM to massively scale the examination of these precious samples, reconstructing 100s of brain cells with and without PHF, tracking axons (and mapping glia and synapses) through much greater brain volumes than was feasible previously. Our goal is to target areas associated with both plaques and tangles, attending to locations where existing findings suggest cell and network vulnerability and contain molecular interactions suspected by some to underlie the initiation and progression of AD. Supporting investigations into the progression of soma/dendritic degeneration, we will target cells operationally defined to represent a progression of neuronal decline as seen in AD; determining the volume fraction of PHF in the cytoplasm as a practical staging measure and linking this to the characterization of quantitative changes in the microstructure of major subcellular constituents. Likewise, we will analyze the progression of axonal degeneration in and near plaques as data obtained suggests that axons may become dystrophic before their parent cell bodies and their dendrites degenerate.